PROJECT SUMMARY Damage to telomeres resulting from radiation or exposure to toxic chemicals can lead to cancer and accelerated aging. Damage may also result from normal metabolism including generation of formaldehyde or transcription when RNA is left behind embedded in the DNA in the form of R-loops. Extensive studies of genomic R-loops have shown them to play both positive roles in regulation of transcription and harmful roles leading to DNA breakage, mutagenesis and cancer. Telomeric R-loops (tel-R-loops) may possibly be the single greatest source of DNA damage at telomeres. Tel-R-loops occur in normal human cells, but are more abundant in cells expressing the ALT cancer phenotype and cells mutated in DNA methylases that produce high levels of telomeric RNA (TERRA). Elevated levels of tel-R-loops have been linked to telomere damage, shortening and high recombination leading to cancer. Radiation, toxic agents such as cisplatin, formaldehyde, and exposure to oxidative damage are also likely to generate higher levels of tel-R-loops. We demonstrated that telomeres are arranged in large loops (t-loops) and recently made a paradigm-shifting discovery linking t-loop formation to telomere transcription which generates TERRA and produces tel-R-loops which we propose are key to t-loop formation. Thus, tel-R-loops are both toxic and necessary for forming protective t-loops. Tel-R-loops are more stable than normal R-loops due to G-quartet formation. The extensive studies of genomic and tel-R-loops have all relied on a single assay employing the S9.6 antibody to DNA/RNA hybrids (DRIP assay). While having driven the field, this IP assay does not discriminate between one or many R-loops on a DNA fragment, or provide information on the clustering of the R-loops, or their size. For tel-R-loops, the IP assay does not reveal whether there are R-loops within the looped portion of the t-loop or their distribution from the sub-telomeric regions to the telomere terminus. For the field to progress, such critical information must be obtained. This can now be done using direct electron microscopic (EM) visualization using methods we have verified and in hand. In the proposed work we will carry out a high resolution study of the large (120-240 nt) particles formed by single stranded G-rich telomeric DNA and TERRA RNA. This is critical for understanding the structure of tel-R- loops and will be done by cryoEM. To determine the frequency, location, size and clustering of tel-R-loops we will apply a novel affinity isolation for telomeric DNA, combined with our battery of EM tools. This will be done using cultured HeLa and human ALT cancer lines and extended to cells treated with toxic chemicals including cisplatin and formaldehyde to introduce crosslinks in the DNA. A novel chemoptogenomic approach for placing ROS generated 8-oxoG lesions specifically at the telomere in cells will be applied in a collaboration and the effect on tel-R-loops determined. The t-loop junction may have important functional roles and this will be explored using assays to detect telomere extension following cleavage of the t-loop junction by HJ resolvases.